Estimated 24-h urinary sodium excretion and risk of end-stage kidney disease

Summary The association between sodium intake and long-term kidney disease endpoints is debated and yet to be proven. We aimed to investigate the associations of estimated 24-h urinary sodium excretion, reflecting daily sodium intake, with the incidence of end-stage kidney disease (ESKD). In this prospective cohort study including 444,375 UK Biobank participant, 865 (0.2%) ESKD events occurred after median follow-up of 12.7 years. For every 1 g increment in estimated 24-h urinary sodium excretion, multivariable-adjusted hazard ratio for incident ESKD was 1.09 (95% confidence interval 0.94–1.26). Nonlinear associations were not detected with restricted cubic splines. The null findings were confirmed by a series of sensitivity analyses, which attenuated potential bias from measurement errors of the exposure, regression dilution, reverse causality, and competing risks. In conclusion, there is insufficient evidence that estimated 24-h urinary sodium excretion is associated with the incidence of ESKD.

The link between sodium intake and end-stage kidney disease (ESKD) is unproven We observed that urinary sodium excretion was not associated with incident ESKD Low sodium intake may not prevent ESKD in a relatively healthy population INTRODUCTION Current guidelines almost uniformly propose low sodium intake in the entire population. [1][2][3] Historically, this recommendation relied on convincing evidence that high sodium intake causally increases blood pressure, as well as on the inference that interventions to reduce blood pressure would subsequently decrease target organ damage. 1 Whether the effects of manipulating sodium intake could successfully translate to the expected benefits remained inconclusive for a long time. 4,5 Until recently, high-quality evidence that ascertains the relationship of sodium intake with cardiovascular events and death was revealed. 6,7 However, somewhat unexpectedly, dietary sodium restriction did not reduce the composite of cardiovascular events in patients with heart failure. 8 Altogether, evidence to date generally supports the benefit of lowering sodium intake on human health, but more efforts should be devoted to a broader population or specific outcomes. 9 Uncontrolled high blood pressure has long been endorsed as an important risk factor for the initiation and progression of kidney diseases. In addition, albuminuria serves as another key determinant of progressive kidney function loss. 10 Akin to its impact on blood pressure, sodium reduction ameliorates urinary excretion of albumin in people with chronic kidney disease (CKD). 11,12 Through causal pathways involving blood pressure and/or albuminuria, excess sodium consumption is thus presumed to drive the long-term progression of kidney impairment. However, robust evidence suggesting low sodium intake delays the progression of kidney function decline is lacking. 9,11,13,14 Previous results of this kind have been mainly obtained in patients with overt CKD. Thus, the association between sodium intake and incident end-stage kidney disease (ESKD) is still debated, particularly in those at low-or moderate-risks. Data from a largescale population-based cohort would be of great value to elucidate the controversy about the hypothesized but unproven benefit of sodium reduction on kidney endpoints.
In this study, we investigated the linear and nonlinear associations of estimated 24-h urinary sodium excretion, reflecting daily sodium intake, with the incidence of ESKD in 444,375 community-dwelling UK Biobank participants. What is more, many cohort studies typically utilizing a single measurement of sodium intake failed to capture the combined effects of its random errors and long-term fluctuations within persons. Such a phenomenon, i.e., regression dilution bias, 15 inevitably introduces downward bias of the estimated association between sodium and diseases. Data on a repeated measurement of urinary biomarkers over years Table 1 shows the baseline characteristics of the study participants collectively, as well as grouped by quartiles of estimated 24-h urinary sodium excretion before the multiple imputations. Among the 444,375 participants, the mean age was 56.2 years, and 54.1% were females. The overall mean estimated 24-h urinary sodium excretion was higher in males than in females (4.0 g versus 2.8 g). In summary, people in the lowest estimated 24-h urinary sodium excretion quartile (Q1) were more likely to be older, white, have a lower Townsend deprivation index, and have a higher education level than those in the highest quartile (Q4). In addition, people in Q1 had a lower likelihood of smoking, alcohol consuming, being obese, and had lower estimated 24-h urinary potassium excretion, blood pressure, and UACR levels. They had a lower prevalence of comorbidities, including hypertension, diabetes, CHD, CHF, and stroke. They are also less likely to be on certain medications, such as diuretics, ACEIs, and ARBs.
The main association results were pooled from the estimates in 5 complete datasets after multiple imputations following Rubin's rule. No multicollinearity was found in all the models presented. The regression dilution ratio (RDR) was 0.84. As shown in Table 2, in crude models, the continuous form and the discrete form of estimated 24-h urinary sodium excretion appeared to be positively associated with incident ESKD. Next, we examined the association between estimated 24-h urinary sodium excretion and incident ESKD, adjusted for potential confounders in Model 1 and 2. Model 1 reached similar conclusions to the crude   Figure 2 was consistent with these results. The HRs for incident ESKD were not significantly different from one, with the confidence intervals overlapping the null across the whole spectrum of estimated 24-h urinary sodium excretion. Also, the restricted cubic spline analysis showed that it did not appear to be any nonlinear relationship with P for nonlinearity of 0.93.

Sensitivity and subgroup analyses
The result that estimated 24-h urinary sodium excretion was not significantly associated with ESKD was supported by sensitivity and subgroup analyses. As shown in Tables S1-S8, Table S9 shows subgroup analysis stratified by sex. The RDR-adjusted HRs (95% CIs) were 0.77 (0.50,1.19) and 1.14 (0.86,1.52) for women and men, respectively. No statistical significance between sex-specific association (p = 0.07) was observed using a 2-sample z-test.

DISCUSSION
In this large prospective population-based cohort study, we observed no linear or nonlinear associations between estimated 24-h urinary sodium excretion and future ESKD in 444,375 UK Biobank participants followed for over 12 years. This finding was confirmed by the correction for regression dilution bias and several sensitivity analyses, which attenuated potential bias from measurement errors and/or long-term intra-individual variability of the exposure, reverse causality, and competing risks. The null results add to the literature that low sodium consumption may not suffice to translate to clinically significant reductions in the risk of ESKD.
Previous cohort study showed that for the population at high risk of future kidney-related events, high urinary sodium excretion was linked with CKD progression and ESKD incidence. He et al. 16 reported a strong association between >4.4 g/d urinary sodium excretion (highest quartile) and CKD progression and allcause mortality in patients with established CKD, in the Chronic Renal Insufficiency Cohort Study. In CKD and general populations, randomized trials have shown that sodium restriction (<2.3 g/d) reduces blood pressure and albuminuria in CKD and general populations, 17-20 without a beneficial effect on kidney function being reported. 18-23 Nevertheless, these interventional studies are typically limited by sample size and short duration. A recent Cochrane systematic review evaluated 21 randomized controlled trials published through October 2020, involving a total of 1,197 adults with CKD. 12 The average study duration was only 7 (range: 1-36) weeks. These studies consequently had to focus on surrogate markers rather than hard endpoints. As a result, intervention studies to date have provided little evidence directly linking iScience Article sodium restriction with long-term kidney outcomes, albeit the findings of lowering blood pressure and albuminuria in the short term are of high certainty.
The results of kidney disease progression therefore have to be largely derived from observational studies. Previous publications, however, show conflicting results investigating the association between sodium intake and subsequent onset or progression of CKD. Several studies observed that high sodium intake was associated with future ESKD, halving of eGFR from baseline, or risk of death, 16,24-28 but the findings could not be replicated by others, 29-31 including a two-sample Mendelian randomization analysis. 32 For this reason, limited and inconsistent evidence supports an association between high sodium intake and kidney outcomes. 11,33 The large discrepancy in findings in the CKD population is attributable, at least to some extent, to reverse causality 34 and measurement errors. 35 Reverse causality, introduced by the fact that overt CKD patients may either eat less or adhere to the recommendation of low sodium intake, tends to lead to underestimation of the sodium-kidney outcome relation. 34 Furthermore, the ability to excrete sodium decreases with progressive decline in kidney function, compromising the reliability of urinary biomarker measurement, which is often utilized as a metric of sodium intake in epidemiological research. 35-Data in the non-CKD population are less prone to these biases. Even though somewhat mixed, 36 most results propose that high sodium intake is associated with developing CKD in high-risk individuals, 37-39 as well as with longitudinal changes in eGFR. 40,41 Nevertheless, the relatively small sample size may have prevented researchers from further determining the association between sodium intake and incident ESKD in the previous literature. 42,43 We found that sodium intake is not a risk factor, or is solely a minor risk factor compared with other established risk factors for incident ESKD. The reasons have not been fully elucidated. One possible interpretation is, as depicted in Figure 3, that sodium restriction leads to a reduction in levels of blood pressure and albuminuria but comes at the cost of higher plasma renin and aldosterone concentrations. Evidence of such iScience Article sideeffects is consistent, indicating a compensatory response to a decrease in effective circulating volume caused by sodium restriction, especially in persons with normal blood pressure. 44 Both renin 45,46 and aldosterone 47 can directly cause kidney damage, in turn counteracting the potential benefits via favorable effects of sodium restriction on blood pressure and albuminuria. Specifically, our study cohort mostly consisted of relatively healthy participants, with approximately 90% and 95% of them being normotensive and normoalbuminuric, respectively. The net effect of sodium intake tended to be neutral after balancing its beneficial and detrimental effects, as observed in such a community-dwelling population.
In conclusion, this population-based cohort study suggests that estimated 24-h urinary sodium excretion is not linearly or nonlinearly associated with the incidence of ESKD. Our finding nonetheless does not support the hypothesized notion that low sodium intake prevents ESKD, at least in individuals without hypertension and albuminuria.

Limitations of the study
The ''gold standard'' method for estimating sodium intake is repeated 24-h urine collection. However, because of the difficulty in such a very large population cohort with multiple study centers and a central biobank, the UK Biobank collected random urinary spot samples. We had employed both the INTERSALT and Kawasaki equations, which led to cross-validation somewhat because the bias of both approaches might not be identical. 48 We could not completely exclude the risk of committing a type II error, particular in the context that another UK Biobank publication did not observe an association of estimated urinary sodium excretion with all-cause mortality and fatal or nonfatal cardiovascular disease events either. 49 Despite the significant associations in model 1, they diminished when anthropometric parameter was additionally included as a covariate in our final models. This might indicate intrinsic pitfalls of the equations we employed. 50 As such, potential methodological limitations need to be considered in interpreting our findings. Nevertheless, the exposure appeared linearly associated with blood pressure and UACR, i.e., positive controls, 17-20 in the current analysis. Furthermore, the use of urinary sodium-to-potassium ratio, exempt from the aforementioned mathematical flaws and also valuable to approximate sodium intake in large surveys, 51 generated results in concert with the main finding. Efforts of this kind might have reduced the probability that exposure misclassification biased our results to the null.
Apart from the lack of measured 24-h urinary sodium excretion, other limitations of our study should also be acknowledged. First, given the observational nature of the data, we described associations rather than iScience Article inferring causality because reverse causality and residual confounding were impossible to completely avoid. Second, participants recruited to the UK Biobank were volunteers and, thus, may not be representative of the general population. In addition, the vast majority of participants were of European white descent despite the inclusion of other ethnicities. Hence, our results should be interpreted cautiously and await confirmation in other ethnic groups with significantly different diets or prevalence of and predispositions to kidney diseases. Lastly, repeat eGFR measures over the follow-up period were not available for most UK Biobank participants, preventing us from further investigating the association of sodium intake with trajectories of kidney function decline.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank all participants, researchers and support staff of the UK Biobank who made the study possible. Y.S. was supported by National Natural Science Foundation of China (82204148) and Scientific Research Figure 3. Proposed interpretation of the net effects of low sodium intake in different people Low sodium intake leads to a reduction in levels of blood pressure and albuminuria but comes at the cost of certain adverse risks, such as higher plasma renin and aldosterone concentrations. In people at high risk, the benefits of low sodium intake exceed the harms; whereas the harms may counteract the benefits in people at low-or intermediate risk (e.g., without hypertension or albuminuria). DBP, diastolic blood pressure; SBP, systolic blood pressure; UACR, urine albumin-to-creatinine ratio.

Study design
The present study is a prospective cohort study based on UK Biobank participants. The study hypothesis arose before inspection of the data. The study hypothesis arose before inspection of the data and a research plan had been submitted to the UK Biobank (application number 73684). However, no protocol for the present analysis was published. Variables used specifically in this work are summarized in Table S10. The process of inclusion and exclusion is depicted in Figure 1. This study included participants with available data on urine sodium and creatinine and without prevalent end-stage kidney disease (ESKD) at baseline according to a prespecified algorithm (https://biobank.ndph.ox.ac.uk/showcase/ukb/docs/ alg_outcome_esrd.pdf, last accessed April 4 th , 2022). The exclusion criteria were incomplete data (age, sex, height, weight, etc.) for estimating 24-h urinary sodium excretion, outliers of estimated 24-h urinary sodium excretion (beyond mean G 5SD), and prevalent malignant tumor at baseline.

Exposure estimates
A spot midstream urine sample was obtained at the end of a 2-h visit and refrigerated between 2 C and 8 C. All urinary biomarkers were measured on a single Beckman Coulter AU5400 clinical chemistry analyzer using the manufacturer's reagents and calibrators, except for urinary albumin, for which reagents and calibrators were sourced from Randox Bioscience. The Beckman Coulter AU5400 analyzer used a potentiometric measurement for the determination of sodium and potassium concentrations and a photometric measurement for the determination of creatinine and albumin concentrations. The analysis method for urinary sodium and potassium involved a sample predilution step, while for urinary albumin and creatinine assays, it allowed samples with results exceeding the upper analytical limit of the assay to be diluted and reanalyzed. Internal quality control was performed for all these urinary biomarkers (https://biobank. ndph.ox.ac.uk/showcase/ukb/docs/urine_assay.pdf, last accessed April 4 th , 2022).
The primary exposure was 24-h urinary sodium excretion (g) estimated from the spot urinary biomarker concentrations based on the sex-specific International Cooperative Study on Salt, Other Factors, and Blood Pressure (INTERSALT) equations with a Western Europe intercept. 53 The INTERSALT equations, adopted in another study based on the UK Biobankdata, 54 are the least biased compared with other predictive equations, including the Kawasaki equations. Unlike the Kawasaki equations, the INTERSALT equations have been developed using nonfasting spot urine samples as obtained in the UK Biobank. We also used the Kawasaki-based estimated 24-h urinary sodium excretion and the urinary sodium-to-potassium ratio as secondary exposures in sensitivity analyses. 55,56 Ascertainment of ESKD

OPEN ACCESS
We used multiple imputation with chained equations to address missing data. The number of multiple imputations was five. The imputation method for continuous data was predictive mean matching (PMM); for binary data was logistic regression (Log-Reg); for unordered categorical data (factor >2 levels) was polytomous regression (PolyReg); and for ordinal data was proportional odds model (Polr). We pooled the estimates from Cox proportional hazards models across imputed datasets using Rubin's rules. 59 The proportional hazards assumption was evaluated using Schoenfeld residuals, and if it is not fulfilled the follow-up time was split accordingly. Multicollinearity was assessed in each model with the variance inflation factor. The variables with variance inflation factor values above 5 were considered to have severe collinearity and were excluded except for one variable that was the most relevant to the outcome in a biological sense.
In addition, we used the McMahon-Peto method to address the regression dilution bias. 15 We calculated estimated 24-h urinary sodium excretion based on the repeated measurements of spot urinary biomarker concentrations. Participants were assigned to quintiles according to the rank of estimated 24-h urinary sodium excretion in the first measurement. The regression dilution ratio was obtained by dividing the difference in the mean estimated 24-h urinary sodium excretion between the 5 th and the 1 st quintiles in the second measurement by the equivalent mean difference in the first measurement. Then, the log(HR)s and standard errors were adjusted by dividing the regression dilution ratios.
We constructed three sequential models. Model 0 was a crude model. Model 1 was adjusted for baseline demographic (age, sex, ethnicity, education, and Townsend deprivation index) and lifestyle factors (smoking, alcohol consumption, physical activity, and estimated 24-h urinary potassium excretion). Model 2 was additionally adjusted for anthropometric measurements (waist circumference), comorbid conditions (hypertension, diabetes, coronary heart disease [CHD], congestive heart failure [CHF], and stroke), medications (diuretics and angiotensin-converting enzyme inhibitors [ACEIs]/angiotensin II receptor blockers [ARBs]), and baseline estimated glomerular filtration rate (eGFR).
We did not adjust for baseline systolic and diastolic blood pressure (SBP/DBP), and urine albumin-to-creatinine ratio (UACR). According to previous literature, 1,10 sodium restriction has been shown to have effects on lowering blood pressure and urinary protein excretion (as stated in Introduction). Therefore, SBP, DBP, and UACR were considered as potential mediators rather than confounders, thus were not adjusted.
We treated hypertension and anti-hypertensive medications as confounders because they represented the condition prior to the baseline. For this reason, the temporal order suggested that they were more likely to be confounders. A history of hypertension and anti-hypertensive medications may affect the urinary sodium excretion estimated at baseline due to the following reasons: 1) some people with existing hypertension may follow medical advice to reduce sodium intake; 2) certain anti-hypertensive medications (eg, diuretics, ACEIs/ARBs) could affect urinary sodium excretion under the pharmacological mechanism. On the other hand, we acknowledge the possibility that some people might have consumed a high amount of sodium